Article
Reference
Mitochondrial calcium handling during ischemia-induced cell death in neurons
GOURIOU, Yves, et al.
Abstract
Mitochondria sense and shape cytosolic Ca(2+) signals by taking up and subsequently releasing Ca(2+) ions during physiological and pathological Ca(2+) elevations. Sustained elevations in the mitochondrial matrix Ca(2+) concentration are increasingly recognized as a defining feature of the intracellular cascade of lethal events that occur in neurons during cerebral ischemia. Here, we review the recently identified transport proteins that mediate the fluxes of Ca(2+) across mitochondria and discuss the implication of the permeability transition pore in decoding the abnormally sustained mitochondrial Ca(2+) elevations that occur during cerebral ischemia.
GOURIOU, Yves, et al . Mitochondrial calcium handling during ischemia-induced cell death in neurons. Biochimie , 2011, vol. 93, no. 12, p. 2060-7
DOI : 10.1016/j.biochi.2011.08.001 PMID : 21846486
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Mini-review
Mitochondrial calcium handling during ischemia-induced cell death in neurons
Yves Gouriou
a, Nicolas Demaurex
a,*, Philippe Bijlenga
b, Umberto De Marchi
aaDepartment of Cell Physiology and Metabolism, University of Geneva, rue Michel-Servet, 1, CH-1211 Genève, Switzerland
bDepartment of Clinical Neurosciences and Dermatology, University of Geneva, Switzerland
a r t i c l e i n f o
Article history:
Received 9 May 2011 Accepted 3 August 2011 Available online 10 August 2011
Keywords:
Calcium signaling Brain diseases Cerebral ischemia Apoptosis Bioenergetics
a b s t r a c t
Mitochondria sense and shape cytosolic Ca2þsignals by taking up and subsequently releasing Ca2þions during physiological and pathological Ca2þelevations. Sustained elevations in the mitochondrial matrix Ca2þconcentration are increasingly recognized as a defining feature of the intracellular cascade of lethal events that occur in neurons during cerebral ischemia. Here, we review the recently identified transport proteins that mediate the fluxes of Ca2þ across mitochondria and discuss the implication of the permeability transition pore in decoding the abnormally sustained mitochondrial Ca2þelevations that occur during cerebral ischemia.
Ó2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Stroke is one of the leading cause of death worldwide and a major cause of long-term disability[1,2]. Stroke can be classified into two major categories: ischemic and hemorrhagic. Ischemic strokes account for around 87% of all strokes[3]and are most frequently the result of the occlusion of a cerebral artery by a blood clot. The current management of stroke involves prevention by treating risk factors surgically (ie: carotid bifurcation sub-occlusions, permeable foramen ovale) or pharmacologically (prescription of acetylsalicylic acid to prevent thrombosis) or rescue attempts by early vessel repermeabilisation (ie: chemical clot dissolution using tissue plas- minogen activator or endovascular mechanical clot removal).
Unfortunately there is no screening to identify a population at risk before thefirst stroke and repermeabilisation is restricted to a short
time window. Thus a majority of patients are still severely disabled or killed by the disease. Despite enormous investments, none of the drug strategies developed to protect ischemic brain tissue have proven to be of any clinical benefit for cardiac arrest or ischemic stroke. The failure has been accounted for by the complex interplay among multiple pathways including excitotoxicity, acidotoxicity, ionic imbalance, oxidative stress, inflammation and apoptosis, which can all lead to cell death and irreversible tissue injury[4]. Brain tissue has a high metabolic rate and thus is particularly vulnerable to ischemic damage. Reduction of the cerebral bloodflow restricts the delivery of oxygen and glucose to the brain tissue and, within minutes, impairs the ability of neurons to maintain ionic gradients [5]. As the cells are unable to maintain a negative membrane potential, neurons depolarize, leading to the opening of voltage- gated calcium channels, and release of excitatory amino acids in the extracellular space. The cascade of events leads to a massive entry of calcium, which is well known to play an essential role in stroke- induced cerebral damage. The increase in free cytosolic calcium is transmitted to the matrix of mitochondria by Ca2þ channels and exchangers located on the inner mitochondrial membrane.
Moderate calcium elevations within the mitochondrial matrix increase the activity of enzymes of the tri-carboxylic cycle, therefore boosting metabolism. Excessive increases in matrix [Ca2þ], however, alter the permeability of mitochondria, impair their ability to generate ATP, and cause the release of pro-apoptotic factors. The mitochondrial dysfunctions resulting from a calcium overload have been shown to be important in the process of ischemia-induced cell death[6]. The role of mitochondrial calcium in neurons in health and disease has been reviewed recently [7] and will only be briefly Abbreviations:AMPAR, 2-amino-3-(3-hydroxy-5-methylisox-azol-4-yl) propio-
nate receptors; ASICS, acid-sensing ion channels; Bcl-2, B-cell lymphoma 2; CaV1.2, L-type voltage-dependent Ca2þchannels; CsA, cyclosporin A; CypD, cyclophilin D;
EF, helixeloopehelix structural domain; IP3, inositol triphosphate receptor; KAR, kainate receptor; Letm1, leucine zipper EF hand containing transmembrane protein 1; MCU, mitochondrial calcium uniporter; MICU1, mitochondrial calcium uptake 1;
MPT, mitochondrial permeability transition; NMDAR, N-methyl-D-aspartate receptor; NCLX, mitochondrial Naþ/Ca2þexchanger; NCX, plasma membrane Naþ/ Ca2þexchangers; PMCA, plasma membrane Ca2þATPase; PTP, permeability tran- sition pore; RNAi, RNA interference; ROS, reactive oxygen species; t-PA, tissue plasminogen activator; TRM2, TRPM7, transient receptor potential ions channels;
UCP2, UCP3, uncoupling protein 2, 3; WHS, Wolf-Hirschhorn syndrome.
*Corresponding author. Tel.:þ41 22 379 5399; fax:þ41 22 379 5338.
E-mail address:Nicolas.Demaurex@unige.ch(N. Demaurex).
Contents lists available atScienceDirect
Biochimie
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b io c h i
0300-9084/$esee front matterÓ2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2011.08.001
mentioned, with a focus on the alterations in mitochondrial calcium homeostasis that occur during ischemia-induced cell death. Here, we will review the transporters involved in the entry and the extrusion of calcium across the inner mitochondrial membrane, and discuss how the discovery of the mitochondrial Ca2þ handling proteins might provide new therapeutic strategies to protect neurons during ischemia.
2. Mechanisms of mitochondrial calcium influx and efflux
Mitochondria contain two membranes, an outer membrane permeable to solutes and an inner membrane impermeable to solutes that harbors the respiratory chain complexes. The respi- ratory chain pumps protons against their concentration gradient from the matrix of the mitochondrion into the inter-membrane space, generating an electrochemical gradient in the form of a negative inner membrane potential and of a pH gradient, the matrix being more alkaline than the cytosol[8,9]. The electrical and chemical components of the proton-motive force add up to energize the back-flux of protons down their electrochemical gradient across the ATP synthase, the enzyme that generates ATP.
The negative m membrane potential (Djm) used to drive the entry of protons also favors the entry of calcium, a divalent cation, into the mitochondrial matrix. As a result, mitochondria can accumu- late large amounts of calcium through a Ca2þ-selective channel known as the mitochondrial Ca2þ uniporter (MCU)[10,11]. The MCU has a relatively low Ca2þ affinity (Kd w10 mM in per- meabilized cells[8]), but Ca2þuptake can be readily detected in intact cells because a significant fraction of mitochondria are located close to calcium release or calcium entry channels and therefore exposed to microdomains of high calcium concentra- tions[12e14]. Electrophysiological recordings of mitoplasts, small vesicles of inner mitochondrial membrane, revealed that the MCU is a highly Ca2þ-selective inward-rectifying ion channel[10]. The activity of the MCU had been known for decades to be inhibited by ruthenium red and its derivative Ru360 [15], but its molecular identity has only been unraveled very recently. In recent years, several molecules have been proposed to be either an essential or an accessory component of the MCU. In 2007, the uncoupling proteins (UCP) 2 and 3[16]were proposed to be essential for the MCU, because overexpression and depletion of these two proteins increased and decreased mitochondrial calcium elevations, respectively, and because mice lacking UCP2 exhibited a reduced sensitivity to the calcium uptake inhibitor ruthenium red.
However, these findings were disputed by another study that reported normal mitochondrial Ca2þ uptake in mice genetically ablated for UCP2 and UCP3[17]. Furthermore, we recently showed that UCP3 modulates the activity of sarco/endoplasmic reticulum Ca2þ ATPases by decreasing mitochondrial ATP production [18].
The mitochondrial Ca2þ alterations associated with changes in UCP3 levels therefore reflect the exposure of mitochondria to abnormal cytosolic Ca2þconcentrations and do not reflect changes in MCU activity. These data indicate that UCP3 is not the mito- chondrial Ca2þ uniporter. In 2009, the leucine zipper EF hand containing transmembrane protein 1(Letm1) [19] was identified by a genome-wide Drosophila RNA interference (RNAi) screen as a molecule that regulate both mitochondrial Ca2þ and Hþ concentrations. Letm1 was reported to be a high-affinity mito- chondrial Ca2þ/Hþexchanger able to import Ca2þat low (i.e. sub- micromolar) cytosolic concentrations into energized mitochon- dria. Earlier studies had however linked Letm1 to mitochondrial potassium/protons exchange and to the maintenance of ionic mitochondrial balance, the integrity of the mitochondrial network and cell viability[20,21]. Importantly, deletion of Letm1 gene has been associated to seizures in Wolf-Hirschhorn syndrome (WHS),
a severe human neurological disease [22]. The high-affinity of Letm1 for Ca2þand its postulated 1Ca2þ/1 Hþstoichiometry are at odds with the known properties of the MCU, and the recent identification of a protein thatfits all MCU properties (see below) indicate that Letm1 is not the dominant mechanism of mito- chondrial Ca2þ uptake. Instead, Letm1 might contribute to an alternate mode of mitochondrial Ca2þ uptake that was first reported in isolated rat liver mitochondria by Gunter’s group.
Using isotopic 45Ca2þmeasurements, these authors showed that the exposure of mitochondria to physiological calcium pulses was sufficient to produce significant mitochondrial Ca2þsequestration via a rapid mode of uptake (RaM). The RaM occurred at the beginning of each pulse and was followed by a slower Ca2þuptake characteristic of the MCU [23]. The RaM was transient and occurred predominantly at Ca2þ concentrations lower than 200 nM, indicating that it was mediated a high-affinity Ca2þ transporter. Subsequent studies usingfluorescent probes reported mitochondrial Ca2þuptake at nanomolar Ca2þconcentrations in a variety of cell types[24,25], and the implications of the coexis- tence of low and high-affinity modes of Ca2þ uptake have been recently reviewed[26]. Interestingly, the RaM can be modeled by a 4 state model whereby Ca2þbinds to an external trigger site to initiate a transient burst of high Ca2þconductivity[27].
In 2010, Palmer and Mootha reported that a new mitochondrial EF hand protein MICU1 (for mitochondrial calcium uptake 1) was required for high capacity mitochondrial calcium uptake, and proposed that MICU acts as a calcium sensor that controls the entry of calcium across the uniporter [28]. Building up on this discovery, two groups simultaneously identified the mitochondrial calcium uniporter in June 2011 [29,30]. Using in silico analysis combined with phylogenetic profiling and analysis of RNA and protein co-expressed with MICU1, the group of Vamsi Mootha isolated a novel protein that co-immunoprecipitated with the exogenously expressed MICU1[30]. Using the same database, the group of Rosario Rizzuto independently identified the same protein. These authors searched for proteins with two or more transmembrane domains (a defining transporter feature) whose expression differed in species known to exhibit or lack uniport activity (kinetoplastids and yeast, respectively)[29]. From the 14 proteins matching these criteria, one contained a highly conserved domain encompassing two transmembrane regions separated by a loop bearing acidic residues, as expected from a mitochondrial Ca2þ channel. Fonctional analysis confirmed that this protein behaves as expected for the mitochondrial uniporter, and it was therefore assigned the defining name of MCU. Mitochondrial Ca2þ uptake was strongly reduced by MCU silencing in cultured cells and in purified mouse liver mitochondria, whereas MCU over- expression enhanced ruthenium red-sensitive mitochondrial calcium uptake in intact and permeabilized cells. The MCU migrated as a large complex of 450 kD on blue native gels, and GFP-tagged MCU could be co-immunoprecipitated by V5-tagged MCU, indicating that the protein forms oligomers [30]. Both studies mapped the MCU to the inner mitochondrial membrane, but disagreed on whether the N and C termini face the matrix[30]
of the inter-membrane space[29]. Mutations of conserved acidic residues within the short sequence linking the two trans- membrane domains abrogated the ability of MCU to reconstitute mitochondrial Ca2þuptake, whereas mutation of a nearby serine resitude (S259) conferred resistance to Ru360, indicating that the acidic residues are required for calcium uptake and that Ser 259 is critical for MCU sensitivity to ruthenium red [30]. Finally, and most convincingly, expression of the purified protein in planar lipid bilayers was sufficient to reconstitute ion channel activity in solutions containing only Ca2þ as the permeant ion [29]. The currents were carried by a channel of small conductance (6e7 pS),
Y. Gouriou et al. / Biochimie 93 (2011) 2060e2067 2061
fast opening/closing kinetics, and low opening probability, and were inhibited by ruthenium red, as expected for the MCU.
Proteins mutated at two of the conserved acidic residues failed to generate Ca2þcurrents when inserted into bilayers and acted as dominant negative when expressed in HeLa cells. These data conclusively show that MCU is the long sought-after mitochon- drial Ca2þuniporter, and open the way to the generation of animal models that will enable to test the role of mitochondrial Ca2þ uptake in cell and tissue physiology. Preliminary experiments are encouraging, as cells overexpressing MCU were more sensitive to apoptosis after treatment with ceramide and H2O2, supporting the notion that mitochondrial Ca2þoverload enhances the sensitivity to apoptosis[29].
Compared to the MCU, the proteins that catalyze the efflux of Ca2þ from mitochondria have received much less attention. The extrusion of Ca2þ from mitochondria is coupled to the entry of sodium across an electrogenic 1Caþ:3Naþexchanger [31] that is inhibited by the benzothiazepine derivative CGP-37157 [32], reviewed in [8]. The subsequent efflux of sodium ions by the mitochondrial 1Naþ:1Hþ exchanger (mNHE)eventually results in the entry of three protons into the matrix for each Ca2þion that leaves mitochondria. Ca2þextrusion thus has a high energetic cost, as it dissipates the proton gradient generated by the respiratory chain that is normally used to drive the synthesis of ATP by the F1F0 ATP synthase (reviewed in [33]). The molecule catalyzing mito- chondrial Naþ/Ca2þexchange has been recently identified as NCLX/
NCKX6, a protein localized in mitochondrial cristae[34], whereas stomatin-like protein 2 (SLP-2), an inner membrane protein, was shown to negatively modulate the activity of the mitochondrial Naþ/Ca2þexchanger [35]. Functional evidence from knock-down and overexpression studies indicate that NCLX is an essential part of the mitochondrial sodium calcium exchanger whereas SLP-2 is an accessory protein that negatively regulates mitochondrial Ca2þ extrusion. The proteins thought to regulate thefluxes of Ca2þacross the inner mitochondrial membrane are summarized inFig. 1, along with commonly used inhibitors of mitochondrial Ca2þ entry and extrusion.
3. Mitochondrial calcium overload during ischemia
During ischemia, neuronal calcium channels and transporters (including NCX, TRM2, TRPM7, ASICS, CaV1.2 and hemichannels) as well as glutamate receptors (NMDAR, AMPAR, KAR) are over- activated, a process known as excitotoxicity (reviewed in [36]).
The increased activity of plasma membrane Ca2þchannels can then trigger the entry of Ca2þ into the cytosol of neurons, leading to larger than usual increases in the cytosolic calcium concentration.
To avoid calcium overload, plasma membrane calcium pumps (PMCA) actively extrude calcium from the cytoplasm during neuronal activity. The increased turnover of PMCA increases the consumption of intracellular ATP that, in neurons, is mainly derived from oxidative phosphorylation occurring in the mitochondria. As discussed above, cytosolic Ca2þelevations are rapidly transmitted to the mitochondrial matrix, where they amplify the activity of Krebs cycle enzymes and of the ATP synthase, thereby increasing the production of ATP[37,38]. During physiological Ca2þelevations, the boost of ATP enables PMCA to extrude the cytosolic calcium and to sustain neuronal activity. During ischemia however, the levels of oxygen and glucose drop rapidly, impairing the production of ATP by mitochondria and by cytosolic glycolysis. As a result, ATP- dependent calcium extrusion mechanisms progressively come to a halt because the intracellular reservoir of ATP is depleted by the continuous activity of the Naþ/KþATPases. The importance of the Naþ/KþATPases in“stealing“ ATP from PMCA could be directly demonstrated as PMCA activity, which collapsed during metabolic
depletion, could be rescued by inhibition of the Naþ/Kþ ATPase [39]. PMCA inhibition amplifies the cytosolic calcium elevations that are transmitted to the mitochondrial matrix, and can then triggers a vicious sequence of mitochondrial calcium overload, mitochondrial dysfunction, release of mitochondrial pro-apotpotic factors, and the activation of death signals[6,40e42]. Mitochondria located near the plasma membrane (subplasmalemmal mitochon- dria) are more exposed to calcium overload due to their proximity to plasma membrane voltage-sensitive calcium channels and to the functionally incapacitated PMCA [43e45]. Subplasmalemmal mitochondria have been shown to take up calcium coming from voltage-gated calcium channels[46], and mitochondrial calcium overload and dysfunction has been linked to glutamate excitotox- icity mediated by the overactivation of NMDA receptors (NMDAs) at the plasma membrane[6,7,47,48]. The specific patterns of cell death triggered by the activation of ionotropic glutamate receptors during excitotoxicity has led to the”route specificity”hypothesis, which postulates that the neurotoxicity depends more on the routes of calcium entry rather than on the magnitude of the calcium over- load[49e51], reviewed in[7]. Intracellular mitochondria are also at risk however, and calcium release from the endoplasmic reticulum has been associated to ischemia induced-cell damage [52e54].
Mitochondria are embedded within sheets of endoplasmic retic- ulum and the two organelles are maintained in very close proximity by linker proteins[55,56]. Because of this proximity, the release of calcium ions through IP3 receptor of the endoplasmic reticulum readily triggers an entry of calcium in adjacent mitochondria [12,13]. Thus, neuronal mitochondria are exposed both to Ca2þions entering across membrane channels and to Ca2þ released from endoplasmic reticulum Ca2þstores. As shown inFig. 2, an elevation in the mitochondrial matrix Ca2þ concentration can be readily recorded in intact PC-12 cells expressing a genetically encoded Ca2þ indicator and exposed to oxygen and glucose deprivation. Although Fig. 1.Ca2þtransport proteins of mitochondria. In mammalian mitochondria, the uptake of Ca2þinto the matrix is mediated by a Ca2þ-selective channel, the mito- chondrial Ca2þuniporter (MCU), regulated by a calcium sensing accessory subunit (MICU1). Letm1 might mediate a slow Ca2þ/Hþexchange at low (nM) cytosolic Ca2þ concentration, driving Ca2þentry, limited by pH gradient. Ca2þis then extruded by the Naþ/Ca2þexchanger NCLX, which is down-regulated by the protein SLP-2. High levels of matrix Ca2þaccumulation trigger the opening of the permeability transition pore (PTP), responsible for mitochondrial membrane permeabilization and neuronal cell death. PTP is also a reversible fast Ca2þrelease channel. The mitochondrial matrix protein cyclophilin D (CypD) facilitates PTP opening by desensitizes PTP to Ca2þ. Common inhibitors of the mitochondrial proteins that control matrix Ca2þuptake and release are indicated in orange.
the magnitude of this ischemia-induced mitochondrial Ca2þ elevation is comparable to the responses evoked by the opening of membrane channels or by the addition of Ca2þ-mobilizing agonists, its duration far exceeds the physiological responses. To our knowledge, such a long-lasting elevation in the mitochondrial matrix free Ca2þconcentration has not been reported, but long- lasting cytosolic Ca2þ elevations are a defining feature of the ischemic process in neurons[57,58]. This Ca2þoverload reflects the failure of Ca2þextruding systems to cope with the excess Ca2þions that enter cells across deregulated plasma membrane channels, following the cleavage of plasma membrane Ca2þpumps[59,60], and Naþ/Ca2þexchangers[61]. As a result of the global cytosolic Ca2þ elevation, mitochondria are exposed to micromolar Ca2þ concentrations for long durations in ischemic neurons, which favors Ca2þuptake by the MCU. The impact of ischemia on the rates of mitochondrial Ca2þuptake is controversial, with some studies reporting decreased mitochondrial Ca2þuptake[62e64], whereas a more recent study reported that 15 min ischemia does not impact the rate of active Ca2þ uptake by isolated mitochondria [65].
Whether the long-lasting Ca2þelevations taking place in the matrix of mitochondria during oxygen and glucose deprivation contribute to the neurotoxicity and can be prevented by MCU inhibition remains to be confirmed.
4. Mitochondrial permeability transition pore and cerebral ischemia
Currently, it is unclear whether the mitochondrial matrix Ca2þ elevations occurring during ischemia are causally related to the neuronal cell death that occurs after cerebral ischemia. The best established link between mitochondrial Ca2þand cellular toxicity is the opening of a Ca2þ-activated channel located in the inner mito- chondrial membrane, the permeability transition pore (PTP) [66e68], responsible for the so-called mitochondrial permeability transition (MPT). In this section we will discuss the molecular nature of PTP, its Ca2þ-dependence and its involvement in cerebral ischemia. The MPT[69]is a phenomenon induced by high levels of matrix Ca2þ accumulation and oxidative stress, responsible for a sudden increase in the mitochondrial inner membrane perme- ability to solutes with molecular masses up to 1500 Da. The MPT plays an important role in events ranging from tissue damage upon infarction to muscle wasting in some forms of dystrophy (see[67]
for a recent review dealing with the involvement of permeability transition in pathological conditions). Importantly, the effect of cyclosporin A (CsA), the most commonly used inhibitor of the MTP, has implicated PTP-dependent mitochondrial dysfunction and Ca2þ deregulation in several diseases, including brain damage following ischemia/reperfusion[70e76]and hypoglycaemia[77]. The PTP is a Ca2þ-, ROS (reactive oxygen species)-, voltage-dependent and Csa- sensitive high-conductance channel, located in the inner mito- chondrial membrane. Despite the great interest generated by this channel, which has been extensively characterized at the pharma- cological and biophysical level, the molecular identity of the PTP is not known. The classical model envisions a supramolecular complex spanning the double membrane system of mitochondria, localized at contact sites [78]. Proteins of all mitochondrial compartments have been proposed to be part of the PTP[66,67,79], in particular cyclophilin D (CypD) in the mitochondrial matrix, the adenine nucleotide translocator in the inner membrane and mitochondrial porin VDAC in the outer membrane. Surprisingly, genetic studies have demonstrated that MPT can still be observed in mitochondria devoid of each of these proteins [80e82]. Some other proteins, including inter-membrane and cytosolic proteins and even the pro- apoptotic Bcl-2 family protein Bax, have been proposed to be part of the PTP under particular conditions [83,84], but Ca2þ-dependent MPT was shown to be independent of Bax[85]. PTP can be regulated by ligands of the outer mitochondrial membrane translocator protein TSPO, formerly known as the peripheral benzodiazepine receptor[86], suggesting that the PTP complex may include TSPO itself [87e90]. Recent results of Sileikyte and collaborators [91]
show that MPT is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO, attesting a regulatory role for this protein. Although the proteins responsible for this important mitochondrial process have not yet been identified, the generation of CypD-knockout mice has now established an unquestionable role for CypD in facilitating PTP opening [80,92e95]. The biophysical properties (conductance, voltage-dependence, selectivity) of PTP were indistinguishable in mitochondria isolated from isogenic wild-type and engineered mice lacking CypD[95], but CsA only inhibited the PTP in wild-type mice [80,95], demonstrating that CypD represents the target for PTP inhibition by CsA. Importantly, mitochondria from CypD-knockout mice displayed a striking desensitization of the PTP to Ca2þ, such that pore opening required about twice the Ca2þload necessary to open the pore in strain-matched, wild-type mitochondria[80].
That the permeability transition is triggered by an elevation in the free Ca2þconcentration within the mitochondrial matrix was discovered early[96e98]. Chelation of matrix Ca2þinduces a rapid closure of PTP and divalent cations such as Mg2þ, Mn2þ, Ba2þand Fig. 2.Changes in mitochondrial matrix [Ca2þ] evoked by oxygen and glucose depri-
vation in PC-12 cells. A. Schematic representation of the genetically encoded“camel- eon”Ca2þindicator D3cpv. The probe consists of two cyan and yellowfluorescent proteins linked by a Ca2þ-sensing module (CaM). Binding of Ca2þto the CaM brings the twofluorescent moieties in closer proximity, favoringfluorescence resonance energy transfer (FRET) and causing a shift influorescence emission from cyan to yellow.
B. Measurement of mitochondrial calcium in PC-12 cells deprived of oxygen and glucose for 30 min and subsequently exposed to an oxygenated solution containing glucose for 15 min. Oxygen was replaced by nitrogen in the environmental chamber.
Mitochondrial calcium rises abruptly upon oxygen/glucose deprivation (OGD), reaches a steady-state plateau, and rapidly returns to basal levels as soon as the normal oxygen tension is restored.
Y. Gouriou et al. / Biochimie 93 (2011) 2060e2067 2063
Sr2þ, instead of inducing PTP opening, can act as inhibitors of Ca2þ trigger sites[99]. The Ca2þ-induced activation of the PTP has been well characterized in mitochondrial patch-clamp experiments[68], the probability of observing PTP activity in mitoplast patches increasing with the increasing [Ca2þ] in mitochondria isolated from mouse, rat or human cells[100]. Moreover, the time required for the channel to inactive was shown to be shorter at lower [Ca2þ], and single channel PTP recordings have demonstrated the competitive nature of the activation by Ca2þand its inhibition by several agents [95,101,102]. Ca2þ-elicited PTP can be pharmaco- logically inhibited and then reactivated by increasing [Ca2þ] for several cycles. This behavior has been suggested to involve an overall equilibrium (instead of a limited number of binding sites on a protein). Cardiolipin, a lipid well-known to bind Ca2þ, has been proposed as“receptor”for this cation in this model[68]. Ca2þis not the only regulator of PTP and many other factors (oxidative stress, voltage, pH, peptides and a wide array of small molecules) are able to modulate the activity of this pore[66,67,69]. Importantly, several regulators of PTP opening act by modulating the Ca2þsensitivity of the pore[66,103]. In addition to being activated by Ca2þ, the PTP has also been proposed to act as a reversible fast Ca2þrelease channel [104]. These functional studies havefirmly established the rela- tionship between [Ca2þ] and the PTP that was subsequently shown to be altered in CypD-knockout mice.
The relationship between MPT and cerebral ischemia inferred from the effects of CsA was nicely confirmed in CypD-knockout mice, by measuring the infarct size after cerebral ischemia/reper- fusion injury induced by the occlusion of the middle cerebral artery [94]. In this study, Schinzel and colleagues demonstrated that iso- lated, CypD-deficent mitochondria showed an increased capacity to retain calcium and were resistant to Ca2þ-induced MPT in swelling experiments. When they induced ischemia/reperfusion, a dramatic decrease in infarct size (62%) was recorded in the brains of CypD- deficient mice, suggesting an essential role for CypD in cell death in the brain. A correlation between gene dosage and the extent of injury was elegantly established, by recording a partial protection in heterozygous mice (37% of reduction in infarct size). These data proved that conditions required for the activation of PTP were present during ischemia/reperfusion, as suggested by earlier pharmacological studies[70,73,74].
An important aspect of the ischemic process is the phenomenon referred as preconditioning, or ischemic tolerance, which offers a therapeutic opportunity to reduce tissue damage after cerebral or cardiac ischemia. This process consists in a short non-injurious ischemic insult that can greatly reduce the severity of a subse- quent prolonged ischemia. This protocol wasfirst described in the dog heart[105], but has since been confirmed in various animal models of brain ischemia [106] and in human stroke patients [107,108]. Different triggers are able to induce preconditioning and several mediating pathways have been characterized, but thefinal effectors remain unknown [108]. Pharmacological evidence [108,109]indicates that the activation of mitochondrial potassium channels might mediate preconditioning by inhibiting MPT acti- vation during reperfusion [109]. Whether mitochondrial ATP- sensitive potassium channels are present in brain and play a role during cerebral ischemia is disputed[108e110], but electrophoretic potassiumflux in brain mitochondria is well established[111]and a voltage-gated potassium channel (Kv1.3) has been electrophysi- ologically characterized in gerbil hippocampal mitochondria[112].
More relevant, two Ca2þ-activated potassium channels (KCa1.1 and KCa3.1) have been detected in inner mitochondrial membrane and characterized by mitochondrial patch-clamp [113,114]. The large conductance Ca2þ-activated potassium channels (BKCaor KCa1.1) is present in mitochondria from glioma cells[113]and from rat brain [115], and has been proposed to contribute to the cardioprotective
effect of potassium influx into mitochondria[116]. Whether the recently discovered mitochondrial intermediate conductance Ca2þ- activated potassium channels (IKCaor KCa3.1)[114]is also present in brain tissue and contributes to the protection against ischemic insults remains to be determined.
Although the causal relationship between mitochondrial Ca2þ accumulation and PTP opening is well established, and despite the fact that MPT invariably leads to neuronal cell death, these rela- tionships do not necessarily imply that matrix Ca2þaccumulation is directly responsible for the injuries related to cerebral ischemia.
The group of Lemasters, for example, has proposed that mito- chondrial Ca2þoverload is a consequence, rather than a cause, of the bioenergetic failure that follows MPT onset. In this view, the mitochondrial Ca2þelevation is only a signature of diseased mito- chondria and is not involved in the induction of the MPT, which occurs after reperfusion[117]. In this study of adult rat myocytes, ROS but not Ca2þoverload has been suggested to trigger pH- and MPT-dependent death after ischemiaereperfusion. Another important parameter to take into account is the timing of the PTP opening during ischemia/reperfusion. In the heart, there is a broad consensus that during ischemia the factors favoring PTP opening (increased matrix Ca2þ and depolarization) are balanced by PTP antagonists (intracellular acidosis, high levels of Mg2þand ADP) that prevent PTP opening during ischemia[118]. Upon reperfusion, oxygen and substrate supplies are restored to the tissue, mito- chondria re-energize, take up the Ca2þthat has accumulated in the cytosol during ischemia, and produce a burst in ROS. The combi- nation of these factors provides ideal conditions for triggering PTP opening[103,118]. Direct methods to asses PTP opening in intact hearts support the concept that PTP is more likely to open upon reperfusion[119,120]. Whether the same sequence of events also occurs in ischemic brain is not known, and further studies are needed to determine the precise timing of the PTP opening during cerebral ischemia. The recent identification of the proteins involved in mitochondrial Ca2þuptake and release provides new opportu- nities to study the role of mitochondrial Ca2þin neuronal death during cerebral ischemia. The role of calcium in MPT activation and cell death can now be directly tested by modulating the expression levels of mitochondrial transport proteins. Targeting the proteins that control the fluxes of Ca2þ should reveal whether altered mitochondrial Ca2þ handling is causally related to ischemic neuronal death, and can potentially increase the repertoire of therapeutic tools to treat ischemic brain diseases.
5. Therapeutic strategies to protect neurons during ischemia by targeting mitochondrial Ca2Dhandling proteins
Patients and therapists alike are eagerly awaiting new strategies allowing brain tissue to survive severe ischemia. Death following ischemia is not a fatality. Some species are remarkably resistant to hypoxia, non excitable cells can be cultured in anaerobic conditions, and various models of brain ischemia as well as human stroke patients’observations confirmed the possibility to greatly reduce the tissue damage after prolonged ischemia by preconditioning [121]. The optimal target to be modulated should be at the convergence of all ischemia signaling pathways but upstream to the irreversible triggers of apoptosis. The mitochondrion is a key organelle in this signaling integration process, and several strate- gies aiming to protect cells from ischemia have therefore focused on mitochondria. Modulation should allow the preservation of the mitochondrial proton gradient and avoid the opening of the mitochondrial permeability transition pore during ischemia and reperfusion. Strategies that target the PTP and its regulation by CypD have been shown to confer significant cardioprotection in isolated rat hearts[122], and the administration of cyclosporine
during percutaneous coronary intervention reduced infarct size in a cohort of patients [123]. Unfortunately cyclosporine causes immunosuppression and nephrotoxicity and the benefits of PTP inhibition are balanced by its adverse effects, as the loss of PTP- mediated Ca2þ efflux increases mitochondrial matrix Ca2þ[124], reviewed in[118]. Inhibition of mitochondrial Ca2þuptake, on the other hand, is expected to reduce the long-lasting mitochondrial calcium elevations that occur during ischemia (Fig. 2) and to prevent PTP opening. The MCU is therefore a prime target as drugs that inhibit this Ca2þuptake system should retain the beneficial effects conferred by PTP inhibition but not its adverse effects.
Accordingly, inhibition of the MCU by ruthenium red protects hearts against ischemia injury[125]. Unfortunately ruthenium red is a very unspecific inhibitor that also inhibit several classes of ion channels and that interfere with the binding of Ca2þto calmodulin (reviewed in[26]). The molecular identification of the MCU opens the way to the rational design of drugs targeting specifically the MCU. Ideally the drug should cross the blood brain barrier and act rapidly within a few seconds. It should be safe enough to be administered preventively when cerebral ischemia is expected during cardiac or cerebro-vascular surgeries, hemorrhagic shock, traumatic brain injuries or any other condition where cerebral bloodflow is compromised. It should be easy and safe to handle to allow very early administration by paramedics when stroke is suspected. Such a medication would substantially increase the fraction of patients that would benefit from curative treatments and reduce disabilities and death.
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